1. Technical Field
The present invention relates to medicine and biomedical research. More specifically, the present invention relates to expression systems to produce small hairpin RNAs (shRNAs) or interfering RNAs (siRNAs), collectively called siRNA in this application, in eukaryotic cells and methods for expressing siRNAs in eukaryotic cells. The present invention also relates to the use of the expression systems as medicinal products.
2. Related Art
RNA interference (RNAi) is a process of sequence-specific, post-transcriptional gene silencing (PTGS) in animals and plants initiated by double-stranded RNA (dsRNA) that is homologous to the silenced gene. It is an evolutionarily conserved phenomenon and a multi-step process that involves generations of active siRNAs in vivo through the action of a mechanism that is not fully understood. RNAi has been used as a reverse genetic tool to study gene function in multiple model organisms, such as plants, Caenorhabditis elegants and Drosophila, where large dsRNAs efficiently induce gene-specific silencing. In mammalian cells dsRNAs, 30 base pairs or longer, can activate antiviral response, leading to the nonspecific degradation of RNA transcripts and to a general shutdown of host cell protein translation. As a result, the long dsRNA is not a general method for silencing specific genes in mammalian cells. Recently, various siRNAs that were synthesized chemically or generated biologically using DNA templates and RNA polymerases have been used to down regulate expression of targeted genes in cultured mammalian cells. Among approaches used, it is highly desirable to use DNA constructs that contain promoters of transcriptions and templates for siRNAs to generate siRNAs in vivo and in vitro. Though several different promoters have been adapted in such DNA constructs, types of promoters used remain limited to, Type III RNA polymerase III (Pol III) promoters, such as the U6 promoter and the H1 promoter, and promoters that require viral RNA polymerases, such as the T7 promoter. The present invention provides methods and designs to produce gene expression suppression agents that greatly expand potential usages of siRNAs.